DNA and protein contents of hepatocytes in primary cultures monitored by flow cytometry: Effect of phenobarbital and dimethylsulphoxide.

ABSTRACT

Primary rat hepatocyte cultures were analysed by two-parameter flow cytometry (FCM) analysis. The simultaneous highly reproducible measurements of DNA and protein contents of hepatocytes showed a high resolution and allowed individual ploidy classes to be monitored during culture. Among both the 2N- and 4N-cell populations of freshly isolated hepatocytes two subpopulations were detectable by their different protein contents. From day 1 to day 7 in culture, the relative contributions of the 2N-, 4N- and 8N-cell populations remained more or less unchanged. The protein content decreased with prolonged culture time to 30% of the original level at day 7. The subpopulations within the 2N- and 4N-cells were no longer detectable. After chronic treatment with phenobarbital (PB, 3 mm), cell detachment was reduced. The contributions of the 2N- and 8N-cell populations decreased whereas that of the 4N-cell population increased. Furthermore, compared to untreated cultures, the cellular protein content was enhanced in all ploidy classes. These PB-induced effects in vitro reflect the hyperplastic and hypertrophic activities of PB that have been observed in vivo. FCM data are compatible with the hypothesis that phenobarbital exerts its tumour promoting activity in vivo by an inhibition of apoptosis. After chronic exposure to dimethylsulphoxide (DMSO, 2%, v/v), a reduction only in the 2N-cell population but an increase in the 8N-cell population was found. The protein content of individual hepatocytes was not enhanced. It seemed that DMSO stabilizes liver cells in vitro at a level that corresponds to the adult stage in vivo. DNA/protein FCM analysis was an efficient tool which was suitable to describe the dynamics of the reactions of hepatocytes in culture after nontoxic, chronic exposure to the growth and differentiation modifying drugs PB and DMSO.